Pacemaker activity, the generation of spontaneous cellular electrical rhythms, governs numerous biological processes from the autonomous beating of the heart to respiratory rhythms and sleep cycles. In the heart, abnormal pacing leads to various forms of arrhythmias (e.g. sick sinus syndrome); If, a diastolic depolarizing current activated by hyperpolarization, is a known key player in cardiac pacing. Despite the fact that If has been recognized for over 20 years, the encoding genes, collectively known as the hyperpolarization-activated cyclic-nucleotide-gated (HCN) or the so-called pacemaker channel family, have been cloned only relatively recently. HCN channels resemble voltage-gated K+ (Kv) channels structurally but two distinct features set them apart physiologically: 1) HCN channels are non-selective (Na+:K+ permeability ratio = 1:4 vs. <1:100 for Kv channels); 2) HCN channels are activated by hyperpolarization rather than by depolarization. The molecular features of these phenotypic differences are unknown. In fact, the ability of lf to drive diastolic depolarization has been questioned because of its extremely slow kinetics and negative activation relative to the time scale and voltage range of cardiac pacing, respectively. The primary objective of this proposal is to improve our understanding of the structure-function relationships of HCN channels, and to examine the physiological relevance of lf in cardiac pacing. To this end, we seek to obtain insights into the signature "backward" gating of HCN channels by characterizing the structural and functional roles of the external S3-S4 linker; to test the "voltage-sensor paddle" and "pore-to-gate" models; to explore the mechanism that couples voltage-sensing to activation by probing the spatial proximity and molecular interactions between the S4 voltage sensor and other neighboring domains; to explore the basis of hysteretic current-voltage behavior of lf; to investigate the physiological roles of native If (and its hysteretic behavior) in cardiac pacing; to modulate the firing rate of endogenous or induced pacemakers. Site-specific amino acid substitutions will be introduced into various regions of the channel based on published work and preliminary data. Channel constructs will be heterologously expressed, followed by detailed characterization using a combination of molecular, biochemical, computational and electrophysiological techniques. Delivery of transgene(s) encoding specific normal or recombinant HCN constructs to cardiomyocytes will be achieved using recombinant adenoviruses. Overall, the proposed work promises not only to improve our understanding of the basic biology of HCN channels but also to provide important insights into the molecular basis of cardiac pacing. Emergent insights may include new approaches to the development of effective therapies for treating abnormalities of sinus node function such as sick sinus syndrome.